Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...

Structural DNA nanotechnology [1,2] and the technique of DNA origami [3] enable the rapid generation of a plethora of complex self-assembled nanostructures. [4][5][6] Since DNA molecules themselves display limited chemical, optical, and electronic functionality, it is of utmost importance to devise methods to decorate DNA scaffolds with functional moieties to realize applications in sensing, catalysis, and device fabrication. Protein functionalization is particulary desirable because it allows exploitation of an almost unlimited variety of functional elements which nature has evolved over billions of years.[7] The delicate architecture of proteins has resulted in no generally applicable method being currently available to selectively couple these components on DNA scaffolds, and thus approaches used so far are based on reversible antibodyantigen interactions, [8,9] aptamer binding, [10,11] nucleic acid hybridization of DNA-tagged proteins, [12,13] or predominantly biotin-streptavidin (STV) interactions. [14][15][16][17][18][19] We demonstrate here that DNA nanostructures can be site-specifically decorated with several different proteins by using coupling systems orthogonal to the biotin-STV system. In particular, benzylguanine (BG) and chlorohexane (CH) groups incorporated in DNA origami have been used as suicide ligands for the site-specific coupling of fusion proteins containing the self-labeling protein tags O 6 -alkylguanine-DNA-alkyltransferase (hAGT), which is often referred to as "Snap-tag", [20] or haloalkane dehalogenase, which is also known as "HaloTag".[21] By using various model proteins we demonstrate the general applicability of this approach for the generation of DNA superstructures that are selectively decorated with multiple different proteins.To realize orthogonal protein immobilization on DNA origami using self-ligating protein tags, we chose the Snap-tag, developed by Johnsson and co-workers, [20] and the commercially available HaloTag [21] system. The respective smallmolecule suicide tags (O 6 -benzylguanine (BG) and 5-chlorohexane (CH)) for both self-labeling protein tags are readily available as amino-reactive N-hydroxysuccinimide (NHS) derivatives (BG-NHS and CH-NHS; Figure 1 a). Complete derivatization of alkylamino-modified oligonucleotides was achieved by coupling with 30 molar equivalents of BG-NHS or CH-NHS, as indicated by electrophoretic analysis (Figure 1 b). To gain access to fusion proteins bearing the complementary Snap-and Halo-protein tags, we constructed expression plasmids by genetic fusion of the genes encoding the protein of interest (POI) and Snap-tag or HaloTag (see the Supporting Information). As model POIs we chose the fluorescent proteins enhanced yellow fluorescent protein (EYFP) [22] and mKate, [23] the enzymes cytochrome C peroxidase (CCP) [24] and esterase 2 from Alicyclobacillus acidocaldarius thermos (EST2), [25] to which the self-labeling tags were fused at the C terminus (POI-Snap or POI-Halo, respectively). In addition, the bispecific Halo-Snap fusion protein...

A DNA-based platform was developed to address fundamental aspects of early stages of cell signaling in living cells. By site-directed sorting of differently encoded, protein-decorated DNA origami structures on DNA microarrays, we combine the advantages of the bottom-up self-assembly of protein-DNA nanostructures and top-down micropatterning of solid surfaces to create multiscale origami structures as interface for cells (MOSAIC). In a proof-of-principle, we use this technology to analyze the activation of epidermal growth factor (EGF) receptors in living MCF7 cells using DNA origami structures decorated on their surface with distinctive nanoscale arrangements of EGF ligand entities. MOSAIC holds the potential to present to adhered cells well-defined arrangements of ligands with full control over their number, stoichiometry, and precise nanoscale orientation. It therefore promises novel applications in the life sciences, which cannot be tackled by conventional technologies.

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